1,4-benzodiazepine-2,5-diones with therapeutic properties

ABSTRACT

The present invention relates to novel chemical compounds, methods for their discovery, and their therapeutic use. In particular, the present invention provides novel 1,4-benzodiazepine-2,5-dione compounds, and methods of using novel 1,4-benzodiazepine-2,5-dione compounds as therapeutic agents to treat a number of conditions associated with the faulty regulation of the processes of programmed cell death, autoimmunity, inflammation, hyperproliferation, and the like.

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 60/732,045, filed Nov. 1, 2005, which is hereinincorporated by reference in its entirety.

This invention was made with government support under AI 47450 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

The present invention relates to novel chemical compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides novel 1,4-benzodiazepine-2,5-dione compounds, andmethods of using novel 1,4-benzodiazepine-2,5-dione compounds astherapeutic agents to treat a number of conditions associated with thefaulty regulation of the processes of programmed cell death,autoimmunity, inflammation, hyperproliferation, vascular abnormalities,cancer, anti-angiogenesis, and the like.

BACKGROUND OF THE INVENTION

Multicellular organisms exert precise control over cell number. Abalance between cell proliferation and cell death achieves thishomeostasis. Cell death occurs in nearly every type of vertebrate cellvia necrosis or through a suicidal form of cell death, known asapoptosis. Apoptosis is triggered by a variety of extracellular andintracellular signals that engage a common, genetically programmed deathmechanism.

Multicellular organisms use apoptosis to instruct damaged or unnecessarycells to destroy themselves for the good of the organism. Control of theapoptotic process therefore is very important to normal development, forexample, fetal development of fingers and toes requires the controlledremoval, by apoptosis, of excess interconnecting tissues, as does theformation of neural synapses within the brain. Similarly, controlledapoptosis is responsible for the sloughing off of the inner lining ofthe uterus (the endometrium) at the start of menstruation. Whileapoptosis plays an important role in tissue sculpting and normalcellular maintenance, it is also the primary defense against cells andinvaders (e.g., viruses) which threaten the well being of the organism.

Not surprisingly many diseases are associated with dysregulation of theprocess of cell death. Experimental models have established acause-effect relationship between aberrant apoptotic regulation and thepathenogenicity of various neoplastic, autoimmune and viral diseases.For instance, in the cell mediated immune response, effector cells(e.g., cytotoxic T lymphocytes “CTLs”) destroy virus-infected cells byinducing the infected cells to undergo apoptosis. The organismsubsequently relies on the apoptotic process to destroy the effectorcells when they are no longer needed. Autoimmunity is normally preventedby the CTLs inducing apoptosis in each other and even in themselves.Defects in this process are associated with a variety of autoimmunediseases such as lupus erythematosus and rheumatoid arthritis.

Multicellular organisms also use apoptosis to instruct cells withdamaged nucleic acids (e.g., DNA) to destroy themselves prior tobecoming cancerous. Some cancer-causing viruses overcome this safeguardby reprogramming infected (transformed) cells to abort the normalapoptotic process. For example, several human papilloma viruses (HPVs)have been implicated in causing cervical cancer by suppressing theapoptotic removal of transformed cells by producing a protein (E6) whichinactivates the p53 apoptosis promoter. Similarly, the Epstein-Barrvirus (EBV), the causative agent of mononucleosis and Burkitt'slymphoma, reprograms infected cells to produce proteins that preventnormal apoptotic removal of the aberrant cells thus allowing thecancerous cells to proliferate and to spread throughout the organism.

Still other viruses destructively manipulate a cell's apoptoticmachinery without directly resulting in the development of a cancer. Forexample, the destruction of the immune system in individuals infectedwith the human immunodeficiency virus (HIV) is thought to progressthrough infected CD4⁺ T cells (about 1 in 100,000) instructinguninfected sister cells to undergo apoptosis.

Some cancers that arise by non-viral means have also developedmechanisms to escape destruction by apoptosis. Melanoma cells, forinstance, avoid apoptosis by inhibiting the expression of the geneencoding Apaf-1. Other cancer cells, especially lung and colon cancercells, secrete high levels of soluble decoy molecules that inhibit theinitiation of CTL mediated clearance of aberrant cells. Faultyregulation of the apoptotic machinery has also been implicated invarious degenerative conditions and vascular diseases.

It is apparent that the controlled regulation of the apoptotic processand its cellular machinery is vital to the survival of multicellularorganisms. Typically, the biochemical changes that occur in a cellinstructed to undergo apoptosis occur in an orderly procession. However,as shown above, flawed regulation of apoptosis can cause seriousdeleterious effects in the organism.

There have been various attempts to control and restore regulation ofthe apoptotic machinery in aberrant cells (e.g., cancer cells). Forexample, much work has been done to develop cytotoxic agents to destroyaberrant cells before they proliferate. As such, cytotoxic agents havewidespread utility in both human and animal health and represent thefirst line of treatment for nearly all forms of cancer andhyperproliferative autoimmune disorders like lupus erythematosus andrheumatoid arthritis.

Many cytotoxic agents in clinical use exert their effect by damaging DNA(e.g., cis-diaminodichroplatanim(II) cross-links DNA, whereas bleomycininduces strand cleavage). The result of this nuclear damage, ifrecognized by cellular factors like the p53 system, is to initiate anapoptotic cascade leading to the death of the damaged cell.

However, existing cytotoxic chemotherapeutic agents have seriousdrawbacks. For example, many known cytotoxic agents show littlediscrimination between healthy and diseased cells. This lack ofspecificity often results in severe side effects that can limit efficacyand/or result in early mortality. Moreover, prolonged administration ofmany existing cytotoxic agents results in the expression of resistancegenes (e.g., bcl-2 family or multi-drug resistance (MDR) proteins) thatrender further dosing either less effective or useless. Some cytotoxicagents induce mutations into p53 and related proteins. Based on theseconsiderations, ideal cytotoxic drugs should only kill diseased cellsand not be susceptible to chemo-resistance.

Many autoimmune diseases and haematologic malignancies result from theaberrant survival and expansion of B and T cells in central andperipheral lymphoid organs. Current therapies for these for thesedisorders generally employ cytotoxic drugs whose mechanisms of actionfrequently involves DNA damage. Hence, the selectivity of these drugs islimited and often relies on the differential ability of diseased andhealthy cells to tolerate and repair drug-induced cellular damage.

What are needed are improved compositions and methods for regulating theapoptotic processes in subjects afflicted with diseases and conditionscharacterized by faulty regulation of these processes (e.g., viralinfections, hyperproliferative autoimmune disorders, chronicinflammatory conditions, and cancers).

SUMMARY

The present invention relates to novel chemical compounds, methods fortheir discovery, and their therapeutic use. In particular, the presentinvention provides novel 1,4-benzodiazepine-2,5-dione compounds, andmethods of using novel 1,4-benzodiazepine-2,5-dione compounds astherapeutic agents to treat a number of conditions associated with thefaulty regulation of the processes of programmed cell death, cancer,anti-angiogenesis, autoimmunity, inflammation, hyperproliferation,vascular abnormalities, and the like. Such compounds and uses aredescribed throughout the present application and represent a diversecollection of compositions and applications.

Certain preferred compositions and uses are described below. The presentinvention is not limited to these particular compositions and uses. Thepresent invention provides a number of useful compositions as describedthroughout the present application.

In certain embodiments, the present invention provides a compositioncomprising novel 1,4-benzodiazepine-2,5-dione compounds. In certainembodiments, the present invention provide a composition comprising acompound described by a formula selected from the group consisting of:

substituted and unsubstituted, including both R and S enantiomeric formsand racemic mixtures.

In some embodiments, R1 is an electron rich heterocycle. In someembodiments, the electron rich heterocycle contains 5 or moreheterocyclic atoms.

In some embodiments, R₁ is selected from the group consisting of

wherein R₁′ is selected from the group consisting of cycloalipathic,aryl, substituted aryl, heterocyclic, and substituted heterocyclic.

In some embodiments, R₂ is selected from the group consisting of H,alkyl, substituted alkyl, and R₁.

In some embodiments, R₃ is selected from the group consisting of H,alkyl, and substituted alkyl.

In some embodiments, R3 is selected from the group consisting ofhydrogen; halogen; OH; a chemical moiety comprising an aryl subgroup; achemical moiety comprising a substituted aryl subgroup; a chemicalmoiety comprising a cycloaliphatic subgroup; a chemical moietycomprising a substituted cycloaliphatic subgroup; a chemical moietycomprising a heterocyclic subgroup; a chemical moiety comprising asubstituted heterocyclic subgroup; a chemical moiety comprising at leastone ester subgroup; a chemical moiety comprising at least one ethersubgroup; a linear or branched, saturated or unsaturated, substituted ornon-substituted, aliphatic chain having at least 2 carbons; a chemicalmoiety comprising Sulfur; a chemical moiety comprising Nitrogen; —OR—,wherein R is selected from the group consisting of a chemical moietycomprising an aryl subgroup; a chemical moiety comprising a substitutedaryl subgroup; a chemical moiety comprising a cycloaliphatic subgroup; achemical moiety comprising a substituted cycloaliphatic subgroup; achemical moiety comprising a heterocyclic subgroup; a chemical moietycomprising a substituted heterocyclic subgroup; a linear or branched,saturated or unsaturated, substituted or non-substituted, aliphaticchain having at least 2 carbons; a chemical moiety comprising at leastone ester subgroup; a chemical moiety comprising at least one ethersubgroup; a chemical moiety comprising Sulfur; a chemical moietycomprising Nitrogen.

In some embodiments, R3 is selected from group consisting of:napthalene; phenol; 1-Napthalenol; 2-Napthalenol;

quinolines, and all aromatic regioisomers.

In some embodiments, the R1 and R3 groups may be interchanged (e.g., insome embodiments, the R1 group is positioned at the first position ofthe benzodiazepine ring and the R3 group is positioned at the thirdposition of the benzodiazepine ring; in some embodiments, the R1 groupis positioned at the third position of the benzodiazepine ring and theR3 group is positioned at the first position of the benzodiazepinering).

In some embodiments, R₄ and R₄′ is independently selected from the groupconsisting of CH₃, halogen, SO₂R₄″, SO₂N(R₄″)₂, OR₄″, N(R₄″)₂,CON(R₄″)₂, NHCOR₄″, NHSO₂R4′, alkyl, mono-substituted alkyl,di-substituted alkyl, tri-substituted alkyl; wherein R₄″ is selectedfrom the group consisting of halogen, H, alkyl, mono-substituted alkyl,di-substituted alkyl, tri-substituted alkyl, aryl, mono-substitutedaryl, di-substituted aryl, tri-substituted aryl, cycloalipathic,mono-substituted cycloalipathic, di-substituted cycloalipathic,tri-substituted cycloalipathic.

In some embodiments, R₅ is selected from the group consisting of H,alkyl, mono-substituted aryl, di-substituted aryl, and tri-substitutedaryl.

In some embodiments, R6 is selected from the group consisting of C, N orS.

In some embodiments, R1 is selected from the group consisting of:

and substituted and unsubstituted, and derivatives thereof.

Certain 1,4-benzodiazepine-2,5-dione compounds of the present inventioninclude, but are not limited to,

In certain embodiments, the present invention provides a method oftreating cells, comprising a) providing i) target cells; and ii) acomposition comprising a 1,4-benzodiazepine-2,5-dione compound having anelectron rich heterocycle at the third carbon position of thebenzodiazepine structure; and b) exposing the target cells to thecomposition under conditions such that said composition interacts withthe target cell so as to induce cellular apoptosis. Such methods finduse in research, drug screening, and therapeutic applications.

In some embodiments, the target cells are in a subject having, forexample, an autoimmune disorder, a haematologic malignancy, or ahyproliferative disorder. In some embodiments, the target cells areselected from the group consisting of in vitro cells, in vivo cells, andex vivo cells. In other embodiments, the target cells are cancer cells.In still other embodiments, the target cells are selected from the groupconsisting of B cells, T cells, and granulocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Bz-423 and an exemplary1,4-benzodiazepine-2,5-dione.

FIG. 2 shows exemplary compounds of the present invention and theirbiological activities.

FIG. 3 shows ATP Synthesis and Hydrolysis Inhibition Graph for1,4-benzodiazepine-2,5-diones.

FIG. 4 shows exemplary compounds of the present invention and theirbiological activities.

FIG. 5 presents additional selectivity data for additional1,4-benzodiazepine-2,5-dione compounds of the present invention.

FIG. 6 shows cellular ATP synthesis in the presence of the compounds ofthe present invention.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used herein, the term “benzodiazepine” refers to a seven memberednon-aromatic heterocyclic ring fused to a phenyl ring wherein theseven-membered ring has two nitrogen atoms, as part of the heterocyclicring. In some aspects, the two nitrogen atoms are in the 1 and 4positions or the 1 and 5 positions, as shown in the general structuresbelow:

The term “larger than benzene” refers to any chemical group containing 7or more non-hydrogen atoms.

The term “chemical moiety” refers to any chemical compound containing atleast one carbon atom. Examples of chemical moieties include, but arenot limited to, aromatic chemical moieties, chemical moieties comprisingSulfur, chemical moieties comprising Nitrogen, hydrophilic chemicalmoieties, and hydrophobic chemical moieties.

As used herein, the term “aliphatic” represents the groups including,but not limited to, alkyl, alkenyl, alkynyl, and acyclic.

As used herein, the term “aryl” represents a single aromatic ring suchas a phenyl ring, or two or more aromatic rings (e.g., biphenyl,naphthalene, anthracene), or an aromatic ring and one or morenon-aromatic rings. The aryl group can be optionally substituted with alower aliphatic group (e.g., alkyl, alkenyl, alkynyl, or acyclic).Additionally, the aliphatic and aryl groups can be further substitutedby one or more functional groups including, but not limited to, chemicalmoieties comprising N, S, O, —NH₂, —NHCOCH₃, —OH, lower alkoxy (C₁-C₄),and halo (—F, —Cl, —Br, or —I).

As used herein, the term “substituted aliphatic” refers to an alkane,alkene, alkyne, or alcyclic moiety where at least one of the aliphatichydrogen atoms has been replaced by, for example, a halogen, an amino, ahydroxy, an ether, a nitro, a thio, a ketone, a sulfone, a sulfonamide,an aldehyde, an ester, an amide, a lower aliphatic, a substituted loweraliphatic, or a ring (aryl, substituted aryl, cycloaliphatic, orsubstituted cycloaliphatic, etc.). Examples of such include, but are notlimited to, 1-chloroethyl and the like.

As used herein, the term “substituted aryl” refers to an aromatic ringor fused aromatic ring system consisting of at least one aromatic ring,and where at least one of the hydrogen atoms on a ring carbon has beenreplaced by, for example, a halogen, an amino, a hydroxy, a nitro, athio, a ketone, an aldehyde, an ether, an ester, an amide, a sulfone, asulfonamide, a lower aliphatic, a substituted lower aliphatic, or a ring(aryl, substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, hydroxyphenyl and thelike.

As used herein, the term “cycloaliphatic” refers to an aliphaticstructure containing a fused ring system. Examples of such include, butare not limited to, decalin and the like.

As used herein, the term “substituted cycloaliphatic” refers to acycloaliphatic structure where at least one of the aliphatic hydrogenatoms has been replaced by a halogen, a heteroatom, a nitro, a thio, anamino, a hydroxy, a ketone, an aldehyde, an ester, an amide, a loweraliphatic, a substituted lower aliphatic, or a ring (aryl, substitutedaryl, cycloaliphatic, or substituted cycloaliphatic). Examples of suchinclude, but are not limited to, 1-chlorodecalyl, bicyclo-heptanes,octanes, and nonanes (e.g., nonrbornyl) and the like.

As used herein, the term “heterocyclic” represents, for example, anaromatic or nonaromatic ring containing one or more heteroatoms. Theheteroatoms can be the same or different from each other. Examples ofheteroatoms include, but are not limited to nitrogen, oxygen and sulfur.Aromatic and nonaromatic heterocyclic rings are well-known in the art.Some nonlimiting examples of aromatic heterocyclic rings includepyridine, pyrimidine, indole, purine, quinoline and isoquinoline.Nonlimiting examples of nonaromatic heterocyclic compounds includepiperidine, piperazine, morpholine, pyrrolidine and pyrazolidine.Examples of oxygen containing heterocyclic rings include, but notlimited to furan, oxirane, 2H-pyran, 4H-pyran, 2H-chromene, andbenzofuran. Examples of sulfur-containing heterocyclic rings include,but are not limited to, thiophene, benzothiophene, and parathiazine.Examples of nitrogen containing rings include, but not limited to,pyrrole, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline,imidazolidine, pyridine, piperidine, pyrazine, piperazine, pyrimidine,indole, purine, benzimidazole, quinoline, isoquinoline, triazole, andtriazine. Nonlimiting examples of heterocyclic rings containing twodifferent heteroatoms include, but are not limited to, phenothiazine,morpholine, parathiazine, oxazine, oxazole, thiazine, and thiazole. Theheterocyclic ring is optionally further substituted with one or moregroups selected from aliphatic, nitro, acetyl (i.e., —C(═O)—CH₃), oraryl groups.

As used herein, the term “substituted heterocyclic” refers to aheterocylic structure where at least one of the ring hydrogen atoms isreplaced by oxygen, nitrogen or sulfur, and where at least one of thealiphatic hydrogen atoms has been replaced by a halogen, hydroxy, athio, nitro, an amino, an ether, a sulfone, a sulphonamide, a ketone, analdehyde, an ester, an amide, a lower aliphatic, a substituted loweraliphatic, or a ring (aryl, substituted aryl, cycloaliphatic, orsubstituted cycloaliphatic). Examples of such include, but are notlimited to 2-chloropyranyl.

As used herein, the term “electron-rich heterocycle,” means cycliccompounds in which one or more ring atoms is a heteroatom (e.g., oxygen,nitrogen or sulfur), and the heteroatom has unpaired electrons whichcontribute to a 6-π electronic system. Exemplary electron-richheterocycles include, but are not limited to, pyrrole, indole, furan,benzofuran, thiophene, benzothiophene and other similar structures.

As used herein, the term “linker” refers to a chain containing up to andincluding eight contiguous atoms connecting two different structuralmoieties where such atoms are, for example, carbon, nitrogen, oxygen, orsulfur. Ethylene glycol is one non-limiting example.

As used herein, the term “lower-alkyl-substituted-amino” refers to anyalkyl unit containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by an amino group. Examplesof such include, but are not limited to, ethylamino and the like.

As used herein, the term “lower-alkyl-substituted-halogen” refers to anyalkyl chain containing up to and including eight carbon atoms where oneof the aliphatic hydrogen atoms is replaced by a halogen. Examples ofsuch include, but are not limited to, chlorethyl and the like.

As used herein, the term “acetylamino” shall mean any primary orsecondary amino that is acetylated. Examples of such include, but arenot limited to, acetamide and the like.

As used herein, the term “a moiety that participates in hydrogenbonding” as used herein represents a group that can accept or donate aproton to form a hydrogen bond thereby. Some specific non-limitingexamples of moieties that participate in hydrogen bonding include afluoro, oxygen-containing and nitrogen-containing groups that arewell-known in the art. Some examples of oxygen-containing groups thatparticipate in hydrogen bonding include: hydroxy, lower alkoxy, lowercarbonyl, lower carboxyl, lower ethers and phenolic groups. Thequalifier “lower” as used herein refers to lower aliphatic groups(C₁-C₄) to which the respective oxygen-containing functional group isattached. Thus, for example, the term “lower carbonyl” refers to interalia, formaldehyde, acetaldehyde. Some nonlimiting examples ofnitrogen-containing groups that participate in hydrogen bond formationinclude amino and amido groups. Additionally, groups containing both anoxygen and a nitrogen atom can also participate in hydrogen bondformation. Examples of such groups include nitro, N-hydroxy and nitrousgroups. It is also possible that the hydrogen-bond acceptor in thepresent invention can be the π electrons of an aromatic ring.

The term “derivative” of a compound, as used herein, refers to achemically modified compound wherein the chemical modification takesplace either at a functional group of the compound (e.g., aromatic ring)or benzodiazepine backbone. Such derivatives include, but are notlimited to, esters of alcohol-containing compounds, esters ofcarboxy-containing compounds, amides of amine-containing compounds,amides of carboxy-containing compounds, imines of amino-containingcompounds, acetals of aldehyde-containing compounds, ketals ofcarbonyl-containing compounds, and the like.

As used herein, the term “subject” refers to organisms to be treated bythe methods of the present invention. Such organisms preferably include,but are not limited to, mammals (e.g., murines, simians, equines,bovines, porcines, canines, felines, and the like), and most preferablyincludes humans. In the context of the invention, the term “subject”generally refers to an individual who will receive or who has receivedtreatment (e.g., administration of a compound of the present inventionand optionally one or more other agents) for a condition characterizedby the dysregulation of apoptotic processes.

The term “diagnosed,” as used herein, refers to the to recognition of adisease by its signs and symptoms (e.g., resistance to conventionaltherapies), or genetic analysis, pathological analysis, histologicalanalysis, and the like.

As used herein, the terms “anticancer agent,” or “conventionalanticancer agent” refer to any chemotherapeutic compounds, radiationtherapies, or surgical interventions, used in the treatment of cancer.

As used herein the term, “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments include, but are not limited to, testtubes and cell cultures. The term “in vivo” refers to the naturalenvironment (e.g., an animal or a cell) and to processes or reactionthat occur within a natural environment.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells,plant cells, fish cells, and insect cells), whether located in vitro orin vivo.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro, including oocytes and embryos.

In preferred embodiments, the “target cells” of the compositions andmethods of the present invention include, refer to, but are not limitedto, lymphoid cells or cancer cells. Lymphoid cells include B cells, Tcells, granulocytes, dendritic cells, and antigen presenting cells.Granulocytes include eosinophils and macrophages. In some embodiments,target cells are continuously cultured cells or uncultured cellsobtained from patient biopsies.

Cancer cells include tumor cells, neoplastic cells, malignant cells,metastatic cells, and hyperplastic cells. Neoplastic cells can be benignor malignant. Neoplastic cells are benign if they do not invade ormetastasize. A malignant cell is one that is able to invade and/ormetastasize. Hyperplasia is a pathologic accumulation of cells in atissue or organ, without significant alteration in structure orfunction.

In one specific embodiment, the target cells exhibit pathological growthor proliferation. As used herein, the term “pathologically proliferatingor growing cells” refers to a localized population of proliferatingcells in an animal that is not governed by the usual limitations ofnormal growth.

As used herein, the term “un-activated target cell” refers to a cellthat is either in the G_(o) phase or one in which a stimulus has notbeen applied.

As used herein, the term “activated target lymphoid cell” refers to alymphoid cell that has been primed with an appropriate stimulus to causea signal transduction cascade, or alternatively, a lymphoid cell that isnot in G_(o) phase. Activated lymphoid cells may proliferate, undergoactivation induced cell death, or produce one or more of cytotoxins,cytokines, and other related membrane-associated proteins characteristicof the cell type (e.g., CD8⁺ or CD4⁺). They are also capable ofrecognizing and binding any target cell that displays a particularantigen on its surface, and subsequently releasing its effectormolecules.

As used herein, the term “activated cancer cell” refers to a cancer cellthat has been primed with an appropriate stimulus to cause a signaltransduction. An activated cancer cell may or may not be in the G₀phase.

An activating agent is a stimulus that upon interaction with a targetcell results in a signal transduction cascade. Examples of activatingstimuli include, but are not limited to, small molecules, radiantenergy, and molecules that bind to cell activation cell surfacereceptors. Responses induced by activation stimuli can be characterizedby changes in, among others, intracellular Ca²⁺, hydroxyl radicallevels; the activity of enzymes like kinases or phosphatases; or theenergy state of the cell. For cancer cells, activating agents alsoinclude transforming oncogenes.

As used herein, the term “effective amount” refers to the amount of acompound (e.g., a compound of the present invention) sufficient toeffect beneficial or desired results. An effective amount can beadministered in one or more administrations, applications or dosages andis not limited intended to be limited to a particular formulation oradministration route.

As used herein, the term “dysregulation of the process of cell death”refers to any aberration in the ability of (e.g., predisposition) a cellto undergo cell death via either necrosis or apoptosis. Dysregulation ofcell death is associated with or induced by a variety of conditions,including for example, autoimmune disorders (e.g., systemic lupuserythematosus, rheumatoid arthritis, myasthenia gravis, Sjögren'ssyndrome, etc.), chronic inflammatory conditions (e.g.,graft-versus-host disease, psoriasis, asthma and Crohn's disease),hyperproliferative disorders (e.g., tumors, B cell lymphomas, T celllymphomas, etc.), viral infections (e.g., herpes, papilloma, HIV), andother conditions such as osteoarthritis and atherosclerosis.

It should be noted that when the dysregulation is induced by orassociated with a viral infection, the viral infection may or may not bedetectable at the time dysregulation occurs or is observed. That is,viral-induced dysregulation can occur even after the disappearance ofsymptoms of viral infection.

A “hyperproliferative disorder,” as used herein refers to any conditionin which a localized population of proliferating cells in an animal isnot governed by the usual limitations of normal growth. Examples ofhyperproliferative disorders include tumors, neoplasms, lymphomas andthe like. A neoplasm is said to be benign if it does not undergo,invasion or metastasis and malignant if it does either of these. Ametastatic cell or tissue means that the cell can invade and destroyneighboring body structures. Hyperplasia is a form of cell proliferationinvolving an increase in cell number in a tissue or organ, withoutsignificant alteration in structure or function. Metaplasia is a form ofcontrolled cell growth in which one type of fully differentiated cellsubstitutes for another type of differentiated cell. Metaplasia canoccur in epithelial or connective tissue cells. A typical metaplasiainvolves a somewhat disorderly metaplastic epithelium.

The pathological growth of activated lymphoid cells often results in anautoimmune disorder or a chronic inflammatory condition. As used herein,the term “autoimmune disorder” refers to any condition in which anorganism produces antibodies or immune cells which recognize theorganism's own molecules, cells or tissues. Non-limiting examples ofautoimmune disorders include autoimmune hemolytic anemia, autoimmunehepatitis, Berger's disease or IgA nephropathy, Celiac Sprue, chronicfatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graftversus host disease, Grave's disease, Hashimoto's thyroiditis,idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis,myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis,scleroderma, Sjorgren syndrome, systemic lupus erythematosus, type 1diabetes, ulcerative colitis, vitiligo, tuberculosis, and the like.

As used herein, the term “chronic inflammatory condition” refers to acondition wherein the organism's immune cells are activated. Such acondition is characterized by a persistent inflammatory response withpathologic sequalae. This state is characterized by infiltration ofmononuclear cells, proliferation of fibroblasts and small blood vessels,increased connective tissue, and tissue destruction. Examples of chronicinflammatory diseases include, but are not limited to, Crohn's disease,psoriasis, chronic obstructive pulmonary disease, inflammatory boweldisease, multiple sclerosis, and asthma. Autoimmune diseases such asrheumatoid arthritis and systemic lupus erythematosus can also result ina chronic inflammatory state.

As used herein, the term “co-administration” refers to theadministration of at least two agent(s) (e.g., a compound of the presentinvention) or therapies to a subject. In some embodiments, theco-administration of two or more agents/therapies is concurrent. Inother embodiments, a first agent/therapy is administered prior to asecond agent/therapy. Those of skill in the art understand that theformulations and/or routes of administration of the variousagents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmfuleffects on a cell or tissue as compared to the same cell or tissue priorto the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to thecombination of an active agent with a carrier, inert or active, makingthe composition especially suitable for diagnostic or therapeutic use invivo, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers toany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions (e.g., such as an oil/wateror water/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants. (See e.g., Martin,Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton,Pa. [1975]).

As used herein, the term “pharmaceutically acceptable salt” refers toany pharmaceutically acceptable salt (e.g., acid or base) of a compoundof the present invention which, upon administration to a subject, iscapable of providing a compound of this invention or an activemetabolite or residue thereof. As is known to those of skill in the art,“salts” of the compounds of the present invention may be derived frominorganic or organic acids and bases. Examples of acids include, but arenot limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric,fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic,toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic,ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic,benzenesulfonic acid, and the like. Other acids, such as oxalic, whilenot in themselves pharmaceutically acceptable, may be employed in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable acid additionsalts.

Examples of bases include, but are not limited to, alkali metals (e.g.,sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides,ammonia, and compounds of formula NW₄ ⁺, wherein W is C₁₋₄ alkyl, andthe like.

Examples of salts include, but are not limited to: acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate,pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like.Other examples of salts include anions of the compounds of the presentinvention compounded with a suitable cation such as Na⁺, NH₄ ⁺, and NW₄⁺ (wherein W is a C₁₋₄ alkyl group), and the like.

For therapeutic use, salts of the compounds of the present invention arecontemplated as being pharmaceutically acceptable. However, salts ofacids and bases that are non-pharmaceutically acceptable may also finduse, for example, in the preparation or purification of apharmaceutically acceptable compound.

As used herein, the term “pathogen” refers a biological agent thatcauses a disease state (e.g., infection, cancer, etc.) in a host.“Pathogens” include, but are not limited to, viruses, bacteria, archaea,fungi, protozoans, mycoplasma, prions, and parasitic organisms.

The terms “bacteria” and “bacterium” refer to all prokaryotic organisms,including those within all of the phyla in the Kingdom Procaryotae. Itis intended that the term encompass all microorganisms considered to bebacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, andRickettsia. All forms of bacteria are included within this definitionincluding cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc.Also included within this term are prokaryotic organisms which are gramnegative or gram positive. “Gram negative” and “gram positive” refer tostaining patterns with the Gram-staining process which is well known inthe art. (See e.g., Finegold and Martin, Diagnostic Microbiology, 6thEd., CV Mosby St. Louis, pp. 13-15 [1982]). “Gram positive bacteria” arebacteria which retain the primary dye used in the Gram stain, causingthe stained cells to appear dark blue to purple under the microscope.“Gram negative bacteria” do not retain the primary dye used in the Gramstain, but are stained by the counterstain. Thus, gram negative bacteriaappear red.

As used herein, the term “microorganism” refers to any species or typeof microorganism, including but not limited to, bacteria, archaea,fungi, protozoans, mycoplasma, and parasitic organisms. The presentinvention contemplates that a number of microorganisms encompassedtherein will also be pathogenic to a subject.

As used herein, the term “fungi” is used in reference to eukaryoticorganisms such as the molds and yeasts, including dimorphic fungi.

As used herein, the term “virus” refers to minute infectious agents,which with certain exceptions, are not observable by light microscopy,lack independent metabolism, and are able to replicate only within aliving host cell. The individual particles (i.e., virions) typicallyconsist of nucleic acid and a protein shell or coat; some virions alsohave a lipid containing membrane. The term “virus” encompasses all typesof viruses, including animal, plant, phage, and other viruses.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of indicating a condition characterized by the dysregulationof apoptotic function may comprise a cell, tissue, or fluids,chromosomes isolated from a cell (e.g., a spread of metaphasechromosomes), genomic DNA (in solution or bound to a solid support suchas for Southern blot analysis), RNA (in solution or bound to a solidsupport such as for Northern blot analysis), cDNA (in solution or boundto a solid support) and the like. A sample suspected of containing aprotein may comprise a cell, a portion of a tissue, an extractcontaining one or more proteins and the like.

As used herein, the terms “purified” or “to purify” refer, to theremoval of undesired components from a sample. As used herein, the term“substantially purified” refers to molecules that are at least 60% free,preferably 75% free, and most preferably 90%, or more, free from othercomponents with which they usually associated.

As used herein, the term “antigen binding protein” refers to proteinswhich bind to a specific antigen. “Antigen binding proteins” include,but are not limited to, immunoglobulins, including polyclonal,monoclonal, chimeric, single chain, and humanized antibodies, Fabfragments, F(ab′)2 fragments, and Fab expression libraries. Variousprocedures known in the art are used for the production of polyclonalantibodies. For the production of antibody, various host animals can beimmunized by injection with the peptide corresponding to the desiredepitope including but not limited to rabbits, mice, rats, sheep, goats,etc. In a preferred embodiment, the peptide is conjugated to animmunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin(BSA), or keyhole limpet hemocyanin [KLH]). Various adjuvants are usedto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacteriumparvum.

For preparation of monoclonal antibodies, any technique that providesfor the production of antibody molecules by continuous cell lines inculture may be used (See e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).These include, but are not limited to, the hybridoma techniqueoriginally developed by Köhler and Milstein (Köhler and Milstein,Nature, 256:495-497 [1975]), as well as the trioma technique, the humanB-cell hybridoma technique (See e.g., Kozbor et al., Immunol. Today,4:72 [1983]), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., in Monoclonal Antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77-96 [1985]).

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778; herein incorporated byreference) can be adapted to produce specific single chain antibodies asdesired. An additional embodiment of the invention utilizes thetechniques known in the art for the construction of Fab expressionlibraries (Huse et al., Science, 246:1275-1281 [1989]) to allow rapidand easy identification of monoclonal Fab fragments with the desiredspecificity.

Antibody fragments that contain the idiotype (antigen binding region) ofthe antibody molecule can be generated by known techniques. For example,such fragments include but are not limited to: the F(ab′)2 fragment thatcan be produced by pepsin digestion of an antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of anF(ab′)2 fragment, and the Fab fragments that can be generated bytreating an antibody molecule with papain and a reducing agent.

Genes encoding antigen binding proteins can be isolated by methods knownin the art. In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.) etc.

As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IbE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin. When a protein orfragment of a protein is used to immunize a host animal, numerousregions of the protein may induce the production of antibodies whichbind specifically to a given region or three-dimensional structure onthe protein; these regions or structures are referred to as “antigenicdeterminants”. An antigenic determinant may compete with the intactantigen (i.e., the “immunogen” used to elicit the immune response) forbinding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “modulate” refers to the activity of a compound(e.g., a compound of the present invention) to affect (e.g., to promoteor retard) an aspect of cellular function, including, but not limitedto, cell growth, proliferation, apoptosis, and the like.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like, that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample (e.g., the level ofdysregulation of apoptosis in a cell or tissue). Test compounds compriseboth known and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by using the screening methods of thepresent invention. A “known therapeutic compound” refers to atherapeutic compound that has been shown (e.g., through animal trials orprior experience with administration to humans) to be effective in suchtreatment or prevention. In preferred embodiments, “test compounds” areagents that modulate apoptosis in cells.

GENERAL DESCRIPTION OF THE INVENTION

As a class of drugs, benzodiazepine compounds have been widely studiedand reported to be effective medicaments for treating a number ofdisease. For example, U.S. Pat. Nos. 4,076,823, 4,110,337, 4,495,101,4,751,223 and 5,776,946, each incorporated herein by reference in itsentirety, report that certain benzodiazepine compounds are effective asanalgesic and anti-inflammatory agents. Similarly, U.S. Pat. No.5,324,726 and U.S. Pat. No. 5,597,915, each incorporated by reference inits entirety, report that certain benzodiazepine compounds areantagonists of cholecystokinin and gastrin and thus might be useful totreat certain gastrointestinal disorders.

Other benzodiazepine compounds have been studied as inhibitors of humanneutrophil elastase in the treating of human neutrophilelastase-mediated conditions such as myocardial ischemia, septic shocksyndrome, among others (See e.g., U.S. Pat. No. 5,861,380 incorporatedherein by reference in its entirety). U.S. Pat. No. 5,041,438,incorporated herein by reference in its entirety, reports that certainbenzodiazepine compounds are useful as anti-retroviral agents.

Despite the attention benzodiazepine compounds have drawn, it willbecome apparent from the description below, that the present inventionprovides novel compounds (e.g., 1,4-benzodiazepine-2,5-dione compounds)and related compounds and methods of using the novel compounds, as wellas known compounds, for treating a variety of diseases.

Benzodiazepine compounds are known to bind to benzodiazepine receptorsin the central nervous system (CNS) and thus have been used to treatvarious CNS disorders including anxiety and epilepsy. Peripheralbenzodiazepine receptors have also been identified, which receptors mayincidentally also be present in the CNS. The present inventiondemonstrates that 1,4-benzodiazepine-2,5-dione compounds with, forexample, an electron rich heterocycle moiety at the C3 position of thebenzodiazepine ring have pro-apoptotic properties consistent with amechanism that does not result from interaction with the mitochondrialF₁F₀-ATPase. The present invention also provides1,4-benzodiazepine-2,5-dione compounds that demonstrate selectivecytotoxicity against T cells as compared to B cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chemical compounds, methods fortheir discovery, and their therapeutic, research, and diagnostic use. Inparticular, the present invention provides 1,4-benzodiazepine-2,5-dionecompounds, and methods of using 1,4-benzodiazepine-2,5-dione compoundsas therapeutic agents to treat a number of conditions associated withthe faulty regulation of the processes of programmed cell death,autoimmunity, inflammation, and hyperproliferation, and the like.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: I. Modulators ofCell Death; II. Exemplary Compounds; III. Pharmaceutical compositions,formulations, and exemplary administration routes and dosingconsiderations; IV. Drug screens; and V. Therapeutic Applications.

The present invention herein incorporates by reference known uses ofbenzodiazepine compounds, including, but not limited to the usesdescribed in Otto, M. W., et al., (2005) J. Clin. Psychiatry 66 Suppl.2:34-38; Yoshii, M., et al., (2005) Nippon Yakurigaku Zasshi125(1):33-36; Yasuda, K. (2004) Nippon Rinsho. 62 Suppl. 12:360-363;Decaudin, D. (2004) 15(8):737-745; Bonnot, O., et al. (2003) Encephale.29(6):553-559; Sugiyama, T. (2003) Ryoikibetsu Shokogun Shirizu.40:489-492; Lacapere, J. J., et al., (2003) Steroids. 68(7-8):569-585;Galiegue, S., et al., (2003) Curr. Med. Chem. 10(16):1563-1572;Papadopoulo, V. (2003) Ann. Pharm. Fr. 61(1):30-50; Goethals, I., etal., (2002) Eur. J. Nucl. Med. Mol. Imaging 30(2):325-328; Castedo, M.,et al., (2002) J. Exp. Med. 196(9):1121-1125; Buffett-Jerrott, S. E., etal., (2002) Curr. Pham. Des. 8(1):45-58; Beurdeley-Thomas, A., et al.,(2000) J. Nuerooncol. 46(1):45-56; Smyth, W. F., et al., (1998)Electrophoresis 19(16-17):2870-2882; Yoshii, M., et al., (1998) NihonShinkei Seishin Yakurigaku Zasshi. 18(2):49-54; Trimble, M. andHindmarch, I. (2000) Benzodiazepines, published by Wrighton BiomedicalPublishing; and Salamone, S. J. (2001) Benzodiazepines and GHB—Detectionand Pharmacology, published by Humana Press.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of organic chemistry, pharmacology,molecular biology (including recombinant techniques), cell biology,biochemistry, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature, such as,“Molecular cloning: a laboratory manual” Second Edition (Sambrook etal., 1989); “Oligonucleotide synthesis” (M. J. Gait, ed., 1984); “Animalcell culture” (R. I. Freshney, ed., 1987); the series “Methods inenzymology” (Academic Press, Inc.); “Handbook of experimentalimmunology” (D. M. Weir & C. C. Blackwell, eds.); “Gene transfer vectorsfor mammalian cells” (J. M. Miller & M. P. Calos, eds., 1987); “Currentprotocols in molecular biology” (F. M. Ausubel et al., eds., 1987, andperiodic updates); “PCR: the polymerase chain reaction” (Mullis et al.,eds., 1994); and “Current protocols in immunology” (J. E. Coligan etal., eds., 1991), each of which is herein incorporated by reference inits entirety.

I. Modulators of Cell Death

In preferred embodiments, the present invention regulates apoptosisthrough the exposure of cells to the compounds of the present invention(e.g., 1,4-benzodiazepine-2,5-diones). In particular, the presentinvention demonstrates that 1,4-benzodiazepine-2,5-diones with, forexample, certain heterocycles at the C3 position of the benzodiazepinering have pro-apoptotic properties consistent with a mechanism that doesnot result from interaction with the mitochondrial F₁F₀-ATPase. Thepresent invention also demonstrates that 1,4-benzodiazepine-2,5-dioneswith an electron rich heterocycle moiety at the C3 position of thebenzodiazepine ring can have pro-apoptotic selectivity for T cells overB cells.

The effect of compounds can be measured by detecting any number ofcellular changes. Cell death may be assayed as described herein and inthe art. In preferred embodiments, cell lines are maintained underappropriate cell culturing conditions (e.g., gas (CO₂), temperature andmedia) for an appropriate period of time to attain exponentialproliferation without density dependent constraints. Cell number and orviability are measured using standard techniques, such as trypan blueexclusion/hemo-cytometry, or MTT dye conversion assay. Alternatively,the cell may be analyzed for the expression of genes or gene productsassociated with aberrations in apoptosis or necrosis.

II. Exemplary Compounds

Exemplary compounds of the present invention are provided below. Certain1,4-benzodiazepine-2,5-dione derivatives have been described (see, e.g.,U.S. patent application Ser. No. 09/700,101; U.S. Pat. No. 6,506,744;Kamal, et al., 2004 Synlett 14:2533-2535; Hulme, et al., 1998 J. Org.Chem. 63:8021-8022; Raboisson et al., 2005 Bioorg. Med. Chem. Lett.15:1857-1861; Raboisson et al., 2005 Bioorg. Med. Chem. Lett.15:765-770; Rabiosson et al., 2005 J. Med. Chem. 48:909-912; each hereinincorporated by reference in their entireties). The present inventionprovides novel 1,4-benzodiazepine-2,5-dione compounds, and uses for1,4-benzodiazepine-2,5-dione compounds.

Certain embodiments provide a composition comprising a compounddescribed by a formula selected from the group consisting of:

In certain embodiments, the present invention provides a compositioncomprising novel 1,4-benzodiazepine-2,5-dione compounds. In certainembodiments, the present invention provide a composition comprising acompound described by a formula selected from the group consisting of:

substituted and unsubstituted, including both R and S enantiomeric formsand racemic mixtures.

In some embodiments, R1 is an electron rich heterocycle. In someembodiments, the electron rich heterocycle contains 5 or moreheterocyclic atoms.

In some embodiments, R₁ is selected from the group consisting of

wherein R₁′ is selected from the group consisting of cycloalipathic,aryl, substituted aryl, heterocyclic, and substituted heterocyclic.

In some embodiments, R₂ is selected from the group consisting of H,alkyl, substituted alky, and R₁.

In some embodiments, R₃ is selected from the group consisting of H,alkyl, and substituted alkyl.

In some embodiments, R3 is selected from the group consisting ofhydrogen; halogen; OH; a chemical moiety comprising an aryl subgroup; achemical moiety comprising a substituted aryl subgroup; a chemicalmoiety comprising a cycloaliphatic subgroup; a chemical moietycomprising a substituted cycloaliphatic subgroup; a chemical moietycomprising a heterocyclic subgroup; a chemical moiety comprising asubstituted heterocyclic subgroup; a chemical moiety comprising at leastone ester subgroup; a chemical moiety comprising at least one ethersubgroup; a linear or branched, saturated or unsaturated, substituted,aliphatic chain having at least 2 carbons; a chemical moiety comprisingSulfur; a chemical moiety comprising Nitrogen; —OR—, wherein R isselected from the group consisting of a chemical moiety comprising anaryl subgroup; a chemical moiety comprising a substituted aryl subgroup;a chemical moiety comprising a cycloaliphatic subgroup; a chemicalmoiety comprising a substituted cycloaliphatic subgroup; a chemicalmoiety comprising a heterocyclic subgroup; a chemical moiety comprisinga substituted heterocyclic subgroup; a linear or branched, saturated orunsaturated, substituted or non-substituted, aliphatic chain having atleast 2 carbons; a chemical moiety comprising at least one estersubgroup; a chemical moiety comprising at least one ether subgroup; achemical moiety comprising Sulfur; a chemical moiety comprisingNitrogen.

In some embodiments, R3 is selected from group consisting of:napthalene; phenol; 1-Napthalenol; 2-Napthalenol;

quinolines, and all aromatic regioisomers.

In some embodiments, the R1 and R3 groups may be interchanged (e.g., insome embodiments, the R1 group is positioned at the first position ofthe benzodiazepine ring and the R3 group is positioned at the thirdposition of the benzodiazepine ring; in some embodiments, the R1 groupis positioned at the third position of the benzodiazepine ring and theR3 group is positioned at the first position of the benzodiazepinering).

In some embodiments, R₄ and R₄′ is independently selected from the groupconsisting of CH₃, halogen, SO₂R₄″, SO₂N(R₄″)₂, OR₄″, N(R₄″)₂,CON(R₄″)₂, NHCOR₄″, NHSO₂R4′, alkyl, mono-substituted alkyl,di-substituted alkyl, tri-substituted alkyl; wherein R₄″ is selectedfrom the group consisting of halogen, H, alkyl, mono-substituted alkyl,di-substituted alkyl, tri-substituted alkyl, aryl, mono-substitutedaryl, di-substituted aryl, tri-substituted aryl, cycloalipathic,mono-substituted cycloalipathic, di-substituted cycloalipathic,tri-substituted cycloalipathic.

In some embodiments, R₅ is selected from the group consisting of H,alkyl, mono-substituted aryl, di-substituted aryl, and tri-substitutedaryl.

In some embodiments, R6 is selected from the group consisting of C, N orS.

In some embodiments, R1 is selected from the group consisting of:

substituted and unsubstituted, and derivatives thereof.

Certain 1,4-benzodiazepine-2,5-dione compounds of the present inventioninclude, but are not limited to,

From the above description, it is apparent that many specific examplesare represented by the generic formulas presented above. A wide varietyof sub combinations arising from selecting a particular group at eachsubstituent position are possible and all such combinations are withinthe scope of this invention. The experimental examples, provided below,describe biological activities of these compounds and provide assays forassessing activities of derivatives or other related compounds.

In summary, a large number of compounds are presented herein. Any one ormore of these compounds can be used to treat a variety of dysregulatorydisorders related to cellular death as described elsewhere herein.Additionally, any one or more of these compounds can be used incombination with at least one other therapeutic agent (e.g., potassiumchannel openers, calcium channel blockers, sodium hydrogen exchangerinhibitors, antiarrhythmic agents, antiatherosclerotic agents,anticoagulants, antithrombotic agents, prothrombolytic agents,fibrinogen antagonists, diuretics, antihypertensive agents, ATPaseinhibitors, mineralocorticoid receptor antagonists, phosphodiesteraseinhibitors, antidiabetic agents, anti-inflammatory agents, antioxidants,angiogenesis modulators, antiosteoporosis agents, hormone replacementtherapies, hormone receptor modulators, oral contraceptives, antiobesityagents, antidepressants, antianxiety agents, antipsychotic agents,antiproliferative agents, antitumor agents, antiulcer andgastroesophageal reflux disease agents, growth hormone agents and/orgrowth hormone secretagogues, thyroid mimetics, anti-infective agents,antiviral agents, antibacterial agents, antifungal agents,cholesterol/lipid lowering agents and lipid profile therapies, andagents that mimic ischemic preconditioning and/or myocardial stunning,antiatherosclerotic agents, anticoagulants, antithrombotic agents,antihypertensive agents, antidiabetic agents, and antihypertensiveagents selected from ACE inhibitors, AT-1 receptor antagonists, ETreceptor antagonists, dual ET/AII receptor antagonists, andvasopepsidase inhibitors, or an antiplatelet agent selected fromGPIIb/IIIa blockers, P2Y₁, and P2Y₁₂ antagonists, thromboxane receptorantagonists, and aspirin) cellular activating agents in along with apharmaceutically-acceptable carrier or diluent in a pharmaceuticalcomposition. The above-described compounds can also be used in drugscreening assays and other diagnostic and research methods.

III. Pharmaceutical Compositions, Formulations, and ExemplaryAdministration Routes and Dosing Considerations

Exemplary embodiments of various contemplated medicaments andpharmaceutical compositions are provided below.

A. Preparing Medicaments

The compounds of the present invention are useful in the preparation ofmedicaments to treat a variety of conditions associated withdysregulation of cell death, aberrant cell growth andhyperproliferation.

In addition, the compounds are also useful for preparing medicaments fortreating other disorders wherein the effectiveness of the compounds areknown or predicted. Such disorders include, but are not limited to,autoimmune disorders disorders. The methods and techniques for preparingmedicaments of a compound of the present invention are well-known in theart. Exemplary pharmaceutical formulations and routes of delivery aredescribed below.

One of skill in the art will appreciate that any one or more of thecompounds described herein, including the many specific embodiments, areprepared by applying standard pharmaceutical manufacturing procedures.Such medicaments can be delivered to the subject by using deliverymethods that are well-known in the pharmaceutical arts.

B. Exemplary Pharmaceutical Compositions and Formulation

In some embodiments of the present invention, the compositions areadministered alone, while in some other embodiments, the compositionsare preferably present in a pharmaceutical formulation comprising atleast one active ingredient/agent, as defined above, together with asolid support or alternatively, together with one or morepharmaceutically acceptable carriers and optionally other therapeuticagents (e.g., a benzodiazepine compound as described in 60/812,270,60/802,394, 60/607,599, and 60/641,040, and U.S. patent application Ser.Nos. 11/445,010, 11/324,419, 11/176,719, 11/110,228, 10/935,333,10/886,450, 10/795,535, 10/634,114, 10/427,211, 10/427,212, 10/217,878,09/767,283, 09/700,101, and related applications; each hereinincorporated by reference in their entireties). Each carrier should be“acceptable” in the sense that it is compatible with the otheringredients of the formulation and not injurious to the subject.

Contemplated formulations include those suitable oral, rectal, nasal,topical (including transdermal, buccal and sublingual), vaginal,parenteral (including subcutaneous, intramuscular, intravenous andintradermal) and pulmonary administration. In some embodiments,formulations are conveniently presented in unit dosage form and areprepared by any method known in the art of pharmacy. Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association (e.g., mixing) the active ingredient withliquid carriers or finely divided solid carriers or both, and then ifnecessary shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tablets,wherein each preferably contains a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. In other embodiments, the activeingredient is presented as a bolus, electuary, or paste, etc.

In some embodiments, tablets comprise at least one active ingredient andoptionally one or more accessory agents/carriers are made by compressingor molding the respective agents. In preferred embodiments, compressedtablets are prepared by compressing in a suitable machine the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder (e.g., povidone, gelatin,hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,disintegrant (e.g., sodium starch glycolate, cross-linked povidone,cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets are made by molding in a suitablemachine a mixture of the powdered compound (e.g., active ingredient)moistened with an inert liquid diluent. Tablets may optionally be coatedor scored and may be formulated so as to provide slow or controlledrelease of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions for topical administration according to thepresent invention are optionally formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. In alternative embodiments, topical formulations comprisepatches or dressings such as a bandage or adhesive plasters impregnatedwith active ingredient(s), and optionally one or more excipients ordiluents. In preferred embodiments, the topical formulations include acompound(s) that enhances absorption or penetration of the activeagent(s) through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethylsulfoxide (DMSO) andrelated analogues.

If desired, the aqueous phase of a cream base includes, for example, atleast about 30% w/w of a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane-1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol and mixturesthereof.

In some embodiments, oily phase emulsions of this invention areconstituted from known ingredients in a known manner. This phasetypically comprises a lone emulsifier (otherwise known as an emulgent),it is also desirable in some embodiments for this phase to furthercomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier so as to act as a stabilizer. In some embodimentsit is also preferable to include both an oil and a fat. Together, theemulsifier(s) with or without stabilizer(s) make up the so-calledemulsifying wax, and the wax together with the oil and/or fat make upthe so-called emulsifying ointment base which forms the oily dispersedphase of the cream formulations.

Emulgents and emulsion stabilizers suitable for use in the formulationof the present invention include Tween 60, Span 80, cetostearyl alcohol,myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

The choice of suitable oils or fats for the formulation is based onachieving the desired properties (e.g., cosmetic properties), since thesolubility of the active compound/agent in most oils likely to be usedin pharmaceutical emulsion formulations is very low. Thus creams shouldpreferably be a non-greasy, non-staining and washable products withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the agent.

Formulations for rectal administration may be presented as a suppositorywith suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, creams, gels, pastes, foams or spray formulations containingin addition to the agent, such carriers as are known in the art to beappropriate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include coarse powders having a particle size, for example, inthe range of about 20 to about 500 microns which are administered in themanner in which snuff is taken, i.e., by rapid inhalation (e.g., forced)through the nasal passage from a container of the powder held close upto the nose. Other suitable formulations wherein the carrier is a liquidfor administration include, but are not limited to, nasal sprays, drops,or aerosols by nebulizer, an include aqueous or oily solutions of theagents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containantioxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents, and liposomes or other microparticulatesystems which are designed to target the compound to blood components orone or more organs. In some embodiments, the formulations arepresented/formulated in unit-dose or multi-dose sealed containers, forexample, ampoules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets of the kind previouslydescribed.

Preferred unit dosage formulations are those containing a daily dose orunit, daily subdose, as herein above-recited, or an appropriate fractionthereof, of an agent.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents. It also is intended that the agents, compositions and methods ofthis invention be combined with other suitable compositions andtherapies. Still other formulations optionally include food additives(suitable sweeteners, flavorings, colorings, etc.), phytonutrients(e.g., flax seed oil), minerals (e.g., Ca, Fe, K, etc.), vitamins, andother acceptable compositions (e.g., conjugated linoelic acid),extenders, and stabilizers, etc.

C. Exemplary Administration Routes and Dosing Considerations

Various delivery systems are known and can be used to administertherapeutic agents (e.g., exemplary compounds as described in Section IIabove) of the present invention, e.g., encapsulation in liposomes,microparticles, microcapsules, receptor-mediated endocytosis, and thelike. Methods of delivery include, but are not limited to,intra-arterial, intramuscular, intravenous, intranasal, and oral routes.In specific embodiments, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion during surgery, injection, or by means of acatheter.

The agents identified can be administered to subjects or individualssusceptible to or at risk of developing pathological growth of targetcells and correlated conditions. When the agent is administered to asubject such as a mouse, a rat or a human patient, the agent can beadded to a pharmaceutically acceptable carrier and systemically ortopically administered to the subject. To identify patients that can bebeneficially treated, a tissue sample is removed from the patient andthe cells are assayed for sensitivity to the agent. Therapeutic amountsare empirically determined and vary with the pathology being treated,the subject being treated and the efficacy and toxicity of the agent.

In some embodiments, in vivo administration is effected in one dose,continuously or intermittently throughout the course of treatment.Methods of determining the most effective means and dosage ofadministration are well known to those of skill in the art and vary withthe composition used for therapy, the purpose of the therapy, the targetcell being treated, and the subject being treated. Single or multipleadministrations are carried out with the dose level and pattern beingselected by the treating physician.

Suitable dosage formulations and methods of administering the agents arereadily determined by those of skill in the art. Preferably, thecompounds are administered at about 0.01 mg/kg to about 200 mg/kg, morepreferably at about 0.1 mg/kg to about 100 mg/kg, even more preferablyat about 0.5 mg/kg to about 50 mg/kg. When the compounds describedherein are co-administered with another agent (e.g., as sensitizingagents), the effective amount may be less than when the agent is usedalone.

The pharmaceutical compositions can be administered orally,intranasally, parenterally or by inhalation therapy, and may take theform of tablets, lozenges, granules, capsules, pills, ampoules,suppositories or aerosol form. They may also take the form ofsuspensions, solutions and emulsions of the active ingredient in aqueousor nonaqueous diluents, syrups, granulates or powders. In addition to anagent of the present invention, the pharmaceutical compositions can alsocontain other pharmaceutically active compounds or a plurality ofcompounds of the invention.

More particularly, an agent of the present invention also referred toherein as the active ingredient, may be administered for therapy by anysuitable route including, but not limited to, oral, rectal, nasal,topical (including, but not limited to, transdermal, aerosol, buccal andsublingual), vaginal, parental (including, but not limited to,subcutaneous, intramuscular, intravenous and intradermal) and pulmonary.It is also appreciated that the preferred route varies with thecondition and age of the recipient, and the disease being treated.

Ideally, the agent should be administered to achieve peak concentrationsof the active compound at sites of disease. This may be achieved, forexample, by the intravenous injection of the agent, optionally insaline, or orally administered, for example, as a tablet, capsule orsyrup containing the active ingredient.

Desirable blood levels of the agent may be maintained by a continuousinfusion to provide a therapeutic amount of the active ingredient withindisease tissue. The use of operative combinations is contemplated toprovide therapeutic combinations requiring a lower total dosage of eachcomponent antiviral agent than may be required when each individualtherapeutic compound or drug is used alone, thereby reducing adverseeffects.

D. Exemplary Co-Administration Routes and Dosing Considerations

The present invention also includes methods involving co-administrationof the compounds described herein with one or more additional activeagents. Indeed, it is a further aspect of this invention to providemethods for enhancing prior art therapies and/or pharmaceuticalcompositions by co-administering a compound of this invention. Inco-administration procedures, the agents may be administeredconcurrently or sequentially. In one embodiment, the compounds describedherein are administered prior to the other active agent(s). Thepharmaceutical formulations and modes of administration may be any ofthose described above. In addition, the two or more co-administeredchemical agents, biological agents or radiation may each be administeredusing different modes or different formulations.

The agent or agents to be co-administered depends on the type ofcondition being treated. For example, when the condition being treatedis cancer, the additional agent can be a chemotherapeutic agent orradiation. When the condition being treated is an autoimmune disorder,the additional agent can be an immunosuppressant or an anti-inflammatoryagent. When the condition being treated is chronic inflammation, theadditional agent can be an anti-inflammatory agent. The additionalagents to be co-administered, such as anticancer, immunosuppressant,anti-inflammatory, and can be any of the well-known agents in the art,including, but not limited to, those that are currently in clinical use.The determination of appropriate type and dosage of radiation treatmentis also within the skill in the art or can be determined with relativeease.

Treatment of the various conditions associated with abnormal apoptosisis generally limited by the following two major factors: (1) thedevelopment of drug resistance and (2) the toxicity of known therapeuticagents. In certain cancers, for example, resistance to chemicals andradiation therapy has been shown to be associated with inhibition ofapoptosis. Some therapeutic agents have deleterious side effects,including non-specific lymphotoxicity, renal and bone marrow toxicity.

The methods described herein address both these problems. Drugresistance, where increasing dosages are required to achieve therapeuticbenefit, is overcome by co-administering the compounds described hereinwith the known agent. The compounds described herein sensitize targetcells to known agents (and vice versa) and, accordingly, less of theseagents are needed to achieve a therapeutic benefit.

The sensitizing function of the claimed compounds also addresses theproblems associated with toxic effects of known therapeutics. Ininstances where the known agent is toxic, it is desirable to limit thedosages administered in all cases, and particularly in those cases weredrug resistance has increased the requisite dosage. When the claimedcompounds are co-administered with the known agent, they reduce thedosage required which, in turn, reduces the deleterious effects.Further, because the claimed compounds are themselves both effective andnon-toxic in large doses, co-administration of proportionally more ofthese compounds than known toxic therapeutics will achieve the desiredeffects while minimizing toxic effects.

IV. Drug Screens

In some embodiments of the present invention, the compounds of thepresent invention, and other potentially useful compounds, are screenedfor pro-apoptotic properties consistent with a mechanism that does notentail interaction with the mitochondrial F₁F₀-ATPase. In preferredembodiments of the present invention, the compounds of the presentinvention, and other potentially useful compounds, are screened forpro-apoptotic selectivity for T cells over B cells.

A number of suitable screens for measuring the binding affinity of drugsand other small molecules to receptors are known in the art. In someembodiments, binding affinity screens are conducted in in vitro systems.In other embodiments, these screens are conducted in in vivo or ex vivosystems.

V. Therapeutic Application

In particularly preferred embodiments, the compositions of the presentinvention are contemplated to provide therapeutic benefits to patientssuffering from any one or more of a number of conditions (e.g., diseasescharacterized by dysregulation of necrosis and/or apoptosis processes ina cell or tissue, disease characterized by aberrant cell growth and/orhyperproliferation, autoimmune diseases, haematologic malignanciesresulting from aberrant survival and expansion of B and T cells incentral and peripheral lymphoid organs, etc.) by modulating (e.g.,inhibiting or promoting) apoptosis in affected cells or tissues. Infurther preferred embodiments, the compositions of the present inventionare used to treat autoimmune/chronic inflammatory conditions.

In particularly preferred embodiments, the compositions of the presentinvention regulate apoptosis through the exposure of cells to thecompounds of the present invention (e.g.,1,4-benzodiazepine-2,5-diones). In particular, the present inventiondemonstrates that 1,4-benzodiazepine-2,5-diones with, for example, anelectron rich heterocycle moiety at the C3 position of thebenzodiazepine ring have pro-apoptotic properties consistent with amechanism that does not result from interaction with the mitochondrialF₁F₀-ATPase. The present invention also demonstrates that1,4-benzodiazepine-2,5-diones with an electron rich heterocycle moietyat the C3 position of the benzodiazepine ring have pro-apoptoticselectivity for T cells over B cells.

Accordingly, preferred methods embodied in the present invention providetherapeutic benefits to patients by providing compounds of the presentinvention that modulate (e.g., inhibiting or promoting) cellularapoptosis in affected cells or tissues without interacting with themitochondrial F₁F₀-ATPase.

In some embodiments, compounds potentially useful in methods of thepresent invention are screened against the National Cancer Institute's(NCI-60) cancer cell lines for efficacy. (See e.g., A. Monks et al., J.Natl. Cancer Inst., 83:757-766 [1991]; and K. D. Paull et al., J. Natl.Cancer Inst., 81:1088-1092 [1989]). Additional screens suitable screens(e.g., autoimmunity disease models, etc.) are within the skill in theart.

In one aspect, derivatives (e.g., pharmaceutically acceptable salts,analogs, stereoisomers, and the like) of the exemplary compounds orother suitable compounds are also contemplated as being useful in themethods of the present invention.

Those skilled in the art of preparing pharmaceutical compounds andformulations will appreciate that when selecting optional compounds foruse in the methods disclosed herein, that suitability considerationsinclude, but are not limited to, the toxicity, safety, efficacy,availability, and cost of the particular compounds.

EXAMPLES

The following examples are provided to demonstrate and furtherillustrate certain preferred embodiments of the present invention andare not to be construed as limiting the scope thereof.

Example 1

This example describes the formulation of exemplary1,4-benzodiazepine-2,5-diones. As shown in FIG. 1, Bz-423 is apro-apoptotic 1,4 benzodiazepine with potent activity in animal modelsof lupus (see, e.g., Blatt, N. B., et al., J. Clin. Invest. 2002, 10,1123; Bednarski, J. J., et al., Arthritis Rheum. 2003, 48, 757; eachherein incorporated by reference in their entireties). Bz-423 binds tothe oligomycin sensitivity conferring protein (OSCP) component of themitochondrial F₁F₀-ATPase (see, e.g., Johnson, K. M., et al., Chemistryand Biology. 2005, 12, 485; herein incorporated by reference in itsentirety). Bz-423 inhibits the enzyme, which produces a state 3 to state4 transition within the mitochondrial respiratory chain (MRC),ultimately resulting in the production of O₂ ⁻ from MRC complex III.This reactive oxygen species functions as a signal—initiating atightly-regulated apoptotic process (see, e.g., Johnson, K. M., et al.,Chemistry and Biology. 2005, 12, 485; herein incorporated by referencein its entirety).

Previous studies revealed that a hydrophobic aromatic substituent at C3along with the phenolic hydroxyl group is required for the cytotoxicactivity of Bz-423. As part of efforts to further define the elements ofBz-423 required for inhibiting the F₁F₀-ATPase, a series of1,4-benzodiazepine-2,5-diones were synthesized as intermediates forother chemistry. Most of these compounds were cytotoxic and unlikeBz-423, many were more selective for T cells compared to B cells (FIG.2). Replacing the napthyl moiety with other hydrophobic groups ofcomparable size (2, 3), but which occupy different space, had relativelysmall effects on activity compared with 1, and in some cases altered theselectivity. By contrast, reducing the size of the C3 group (4, 5) wasnot tolerated. Given the similarity between the structures in FIG. 2 andthe pro-apoptotic 1,4-benzodiazepines previously reported (see, e.g.,Johnson, K. M., et al., Chemistry and Biology. 2005, 12, 485; hereinincorporated by reference in its entirety), experiments were conductedto determine if the 1,4-benzodiazepine-2,5-dione compounds inhibit theF₁F₀-ATPase and generate O₂ ⁻ in the same manner as Bz-423. Compoundslisted in FIG. 2 neither blocked the F₁F₀-ATPase nor inhibited by agentsthat specifically scavenge superoxide. These results indicated that the1,4-benzodiazepine-2,5-dione compounds shown in FIG. 2 have a differentmolecular target and apoptotic mechanism than Bz-423. By contrast, them-biphenyl analog inhibited ATP hydrolysis while not blocking synthesis,similar to previously reported 1,4-benzodiazepine (see, e.g., Hamann, L.G., et al., Bio. Med. Chem. Lett. 2004, 14, 1031; herein incorporated byreference in its entirety).

Example 2

This example describes the optimization of the novel1,4-benzodiazepine-2,5-dione compounds of the present invention. Thedata in FIG. 2 show that the size of the C3 is important for theactivity of the 1,4-benzodiazepine-2,5-dione compounds. Moreover, thesedata suggest that it is possible to optimize potency and selectivitybased on the biphenyl or 2-napthylene C3 side chains. A range ofsubstituted biphenyls can be prepared readily by Suzuki couplings ofaryl halides with commercially available boronic acids (see, e.g.,Suzuki, A. Acc. Chem. Res. 1982, 15, 178; herein incorporated byreference in its entirety). Therefore, the relationship between thestereoelectronics of the C3 side chain and cytotoxicity of the1,4-benzodiazepine-2,5-dione compounds, was further evaluated bysynthesizing substituted analogs of 10 (FIG. 3).

In the first group of derivatives a methyl group or chlorine atom wassubstituted at each of the 2′, 3′, or 4′-positions. Analysis of thesecompounds revealed that substitution had little effect of killing Tcells but improved the potency against B cells. Since substitution ateither the 3′ or 4′ position led to the greatest improvement in potency,substitutions at those positions was further explored. The addition ofelectron rich substituents to the meta and para positions (26, 27)increased potency, whereas the carboxylic acid 28 had the reverseeffect. In addition, electron rich, heterocyclic aromatic rings (30-33)provided selectively potent compounds, namely (30, 31). Collectively,this data indicate that electron rich aromatic heterocycles at R₁ ofFIG. 4 provide optimal activity and selectivity, although the presentinvention is not limited to such compounds.

FIG. 5 presents additional selectivity data for additional1,4-benzodiazepine-2,5-dione compounds of the present invention. RamosEC50 refers to the concentration of drug required to 50% of Ramos Bcells and Jurkat EC50 refers to the concentration of drug required to50% of Jurkat T cells. Selectivity was calculated by dividing the B cellEC50 data by the that for the T cells. All measurements were conductedas described previously (see, e.g., T. Francis, et al., Bioorg. Med.Chem. Lett. 2006 16, 2423-2427; herein incorporated by reference in itsentirety).

Example 3

This example demonstrates that 1,4-benzodiazepine-2,5-dione compounds ofthe present invention do not inhibit ATP synthesis. The following fourcompounds were exposed to cells, and ATP synthesis measured:

As shown in FIG. 6, the following compounds

failed to inhibit ATP synthesis, while

did inhibit ATP synthesis.

All publications and patents mentioned in the above specification areherein incorporated by reference. Although the invention has beendescribed in connection with specific preferred embodiments, it shouldbe understood that the invention as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the relevant fields are intended to be within the scope ofthe following claims.

1. A compound represented by the following formula:

including R and S enantiomeric forms, racemic mixtures, andpharmaceutically acceptable salts thereof, wherein: R₁ is

R₂ and R₅ are hydrogen; R₃ is hydrogen or alkyl; and R₄ is halogen. 2.The compound of claim 1, wherein said compound is selected from thegroup consisting of:


3. The compound of claim 1, wherein said compound is represented by:

or a pharmaceutically acceptable salt thereof.
 4. The compound of claim1, wherein said compound is represented by:

or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising a compound of claim 1 and a pharmaceuticallyacceptable carrier.
 6. A method of treating a disorder selected from thegroup consisting of graft-versus-host disease, rheumatoid arthritis,Sjogren's syndrome, myasthenia gravis, asthma, psoriasis, cancer,systemic lupus erythematosus, multiple sclerosis, celiac sprue,idiopathic thrombocytopenia purpura, scleroderma, Crohn's Disease,inflammatory bowel disease, and ulcerative colitis, comprisingadministering an effective amount of a compound of claim 1 to a patientin need thereof to treat said disorder.
 7. The method of claim 6,wherein the disorder is graft-versus-host disease or psoriasis.
 8. Themethod of claim 6, wherein the disorder is rheumatoid arthritis.
 9. Themethod of claim 6, wherein the disorder is Crohn's Disease, inflammatorybowel disease, or ulcerative colitis.
 10. The method of claim 6, whereinthe disorder is cancer.
 11. The method of claim 6, wherein said compoundis represented by:

or a pharmaceutically acceptable salt thereof.
 12. The method of claim7, wherein said compound is represented by:

or a pharmaceutically acceptable salt thereof.
 13. The method of claim8, wherein said compound is represented by:

or a pharmaceutically acceptable salt thereof.
 14. The method of claim9, wherein said compound is represented by:

or a pharmaceutically acceptable salt thereof.